chapter 3. _environmental Biology_

Environmental Biology

BIOLOGICAL EFFECTS OF WEIGHTLESSNESS AND ZERO GRAVITY

High priority has been given to studies of weightlessness. Gravity is one of the most fundamental forces that acts on living organisms, and all life on Earth except the smallest appears to be oriented with respect to gravity, although certain organisms are more responsive to it than others. The gravity force on Earth is 1 g, but this force may be experimentally varied from zero g, or weightlessness, to many thousands of g’s.

Zero gravity or decreased gravity occurs during freefall, in parabolic trajectory, or during orbit around the Earth. Gravitational force decreases by the square of the distance away from the Earth’s center. It is reduced about 5 percent at about 200 nautical miles’ altitude. Gravitational force greater than 1 g can be obtained by acceleration, deceleration, or impact. It also can be increased by using a centrifuge which adds a radial acceleration vector to the 1 g of Earth.

On the ground, the biological effects of gravity have been studied at 1 g, and experimentally, forces of many g have been produced. In addition, modifications of the effects of the 1-g force have been induced by suspension of the organism in water or by horizontal immobilization of an erect animal such as man. The biological effects of such modification have been of significant value in understanding some of the possible consequences of human exposure to the zero-g environment of space.

Weightlessness in an Earth-orbiting satellite occurs when the continuous acceleration of Earth’s gravity is exactly counterbalanced by the continuous radial acceleration of the satellite. In such a weightless state, organisms are liberated from their natural and continuous exertion against 1 g, but this liberation may carry with it certain serious physical penalties.

Some of the physical processes which probably have the greatest biological effects are (1) convective flow of fluid, e.g., protoplasmic streaming, transport of nutrient materials, oxygen, waste products, and CO₂ from the immediate environment of the cell, and (2) sedimentation occurring within cells; substances of higher density sediment in a gravitational field, and those of lighter density rise. A separation of particles of different densities probably occurs. The removal of gravity would change a distribution of particles like mitochondria by 10 percent ([ref.64]).

Gravity has effects on the physical processes involved in mitosis and meiosis. Study under weightlessness might contribute to our understanding of the general cellular information-relay process.

A gravitational effect is known in the embryonic development of the frog Rana sylvatica. After fertilization, the eggs rotate in the gravitational field so that the black animal hemisphere is uppermost. Development becomes abnormal if this position is disturbed. If the egg is inverted following the first cleavage and held in this position, two abnormal animals result, united like Siamese twins. This phenomenon appears to be related to the gravitational separation of low- and high-density components of the egg. The size of the egg is about 1 to 2 mm and is suspended in water of about the same density. This system is very sensitive to gravity; and, under weightlessness, the separation of different density components might be irregular, leading to aberrant development. When certain aquatic insect eggs are inverted, subsequent development results in shortened abnormal larvae.

The directional growth of plant shoots and plant roots is probably due to this sedimentation phenomenon, particularly the effect on movement of auxins ([ref.65]).

Free convection flow is a major transport process, and under its influence the mixing of substances is much more effective than when diffusion operates alone. Free convection flow is a macroscopic phenomenon which increases not only with g, but varies also approximately with the five-fourths power of the bulk concentration involved. Whether or not convection is important at the microscopic level remains an experimentally unsolved question. The Grashoff number limits free convection to the macroscopic domain. It would appear in weightlessness that the contribution of free convective flow would be small and that only diffusion should occur. This phenomenon would cause equilibration to occur much more slowly than that occurring with free convection and diffusion. The absence of convective transfer raises a problem as to how nutrients may be obtained and waste products removed in living cells during weightlessness. In a liquid substrate, nutrients and oxygen would be depleted, and waste products would accumulate around the cell.

Absence of gravity may have far-reaching consequences in the homeostatic aspects of cell physiology. The outstanding characteristics of living cells which are most likely to be influenced by the absence of gravity are the ability of the cell to maintain its cytoplasmic membrane in a functional state, the capacity of the cell to perform its normal functions during the mitotic cycle, and the capacity of the cytoplasm to maintain the constant reversibility of its sol-gel system ([ref.66]).

Two-phase systems, e.g., air-in-water and air-in-oil, possess entirely different characteristics at zero g than at 1 g. These physical differences in phase interaction could well be suspected of interfering with the orientation and flow pattern of cell constituents, thus hindering the cellular processes involved in the movement, metabolism, and storage of nutrients and waste.

On the basis of theoretical calculations, weightlessness can be expected to have some effect even on one individual cell if its size exceeds 10 microns in diameter ([ref.64]). Cell colonies might be affected. In larger cells there may be a redistribution of enzyme-forming systems which give rise to polarization. The low surface tension of the cell membrane lends itself to hydrostatic stress distortion, implying an alteration in permeability and thus an almost certain alteration of cell properties under low gravity conditions.

Another aspect of gravity that affects the growth and development of living organisms is the directionality of the gravitational field. In fact, some plants are so sensitive that they are able to direct their growth with as little stimulus as a 1×10⁻⁶ gravitational field. Investigations of plant growth in altered gravitational fields are underway at Argonne National Laboratory and Dartmouth College.

The Argonne Laboratory has designed and developed a 4-pi, or omnidirectional, clinostat. By rotating a plant so that the force of gravity is distributed evenly over all possible directions, the directional effects of gravity are eliminated, simulating some aspects of the zero-g state. It was shown that certain plants grew more slowly and had fewer and smaller leaves, while others had about 25 percent greater replication of fronds and had greater elongation of certain plant parts. It will be extremely interesting to compare these effects under zero-g conditions in orbiting spacecraft.

The effect of gravity in transporting growth hormones in plants has been demonstrated at Dartmouth College using radiocarbon-labeled growth hormones. Plant geotropisms and growth movements have been studied and biosatellite experiments developed.

Anatomy is considered a derivative adaptation to gravity ([ref.67]). A large background of plant research exists on the effect of orientation on plant responses. Information from clinostat experiments is considered susceptible of extrapolation to low gravity conditions because the threshold period for gravitational triggering is relatively long.

Once over critical minimum dimensions, the major effects of low gravity would be assumed to occur in those heterocellular organisms that develop in more or less fixed orientation with respect to terrestrial gravity and which respond to changes in orientation with relatively long induction periods; these are the higher plant orders. On the other extreme are the complex primates which respond rapidly, but whose multiplicity of organs and correlative mechanisms are susceptible to malfunction and disorganization. It may be suggested that the heterocellular lower plants and invertebrates will be less affected. Perturbations of the environment to which the experimental organism is exposed must be limited or controlled to reduce uncertainties in interpretation of the results. At the same time, the introduction of known perturbations may assist in isolating the effects due solely to gravity. Study of de novo differentiation and other phenomena immediately after syngamy may be of particular importance. Study of anatomical changes after exposure of the organism to low gravity is important.

BIOLOGICAL EFFECTS OF SPACE RADIATION¹

¹ This section includes part of the Summary of the Panel on Radiation Biology of the Environmental Biology Committee Space Science Board, NAS/NRC (1963), and results of research by the Bioscience Programs, NASA.

Radiation sources in space are of three types: galactic cosmic radiation, Van Allen belts, and solar flares with an intense proton flux. Cosmic radiation has higher energy levels than radiation produced by manmade accelerators.

The Panel on Radiation Biology, while recognizing the need for radiobiological studies of an applied nature with reference to manned flight programs, stated that it would be shortsighted for the United States to confine its efforts to the solution of immediate problems since, in the long run, successful exploration of space will be aided by the contributions of basic research. Both the immediate biological research program and the continuing program for basic studies should be built upon the large body of existing knowledge of radiation effects. The attitude that all radiobiological experiments need be repeated in the space environment should be resolutely rejected. Since fundamental radiobiology cannot be performed easily in space, it has been recommended that, wherever possible, these investigations be carried out in ground laboratories in preference to flying laboratories.

Space environment does vary from the terrestrial environment, but the variations are not so great as to lead to the expectation of strikingly different biological effects of radiation in space. However, it is conceivable that radiations whose effects are well known under terrestrial conditions may have some unsuspected biological effects when combined with unusual features of the space environment: e.g., zero g. Previous space radiobiological studies have depended solely on very low and inaccurately measured doses of ambient space radiation. It has been difficult to distinguish between the observed response levels and the random noise; thus, experiments have been inconclusive.

Biological Effects of Heavy Ions and Mesons

The biological effects of heavy ions (especially Z>2) and mesons are of specific interest to space radiobiology.

Controlled Radiobiological Experiments in Space

There is the remote possibility that the radiobiological response may be modified by factors as yet unknown and perhaps not susceptible to terrestrial study. Experiments have been designed to settle this matter including the exposure of biological materials during space flight which meet the following criteria of reliability: (1) the use of well-known biological systems, e.g., mutation induction or chromosome breakage; (2) the use of a sufficient number of individuals in the experiment to guarantee statistical precision on the results; (3) the exposure of the system to known quantities and qualities of radiation; (4) the use of adequate controls.

High-altitude balloon ascents of the 1930’s initiated study of the biological effects of cosmic rays. They were limited to the exploration of secondary cosmic radiation effects. After World War II, the research extended to the use of V-2 rockets fired from the White Sands Proving Ground. Interest returned to balloons and a significant program was underway by 1950, first using mice and then hamsters, fruit flies, cats, and dogs. These flights gave no evidence of radiation damage. However, it was realized that the flights were too far south to obtain a significant exposure, and more northerly flights began in 1953. Mice and guinea pigs were flown on these later flights. Chase ([ref.68]) showed the most unequivocal results to that time, a statistically significant increase in light hairs on black animals and the streaks of white hair up to 10 times wider than expected. Brain lesions were detected in the guinea pigs flown on Man High in 1957. Many other types of biological material were sent aloft in an effort to further corroborate existing information and to investigate genetic and developmental effects of cosmic radiation.

From the earlier V-2 rocket flights to the Jupiter missile launchings of the monkeys Able and Baker, cosmic-ray research was continued, but the short flight durations of these vehicles did not provide substantial information. The USAF Discoverer satellite program has given impetus to cosmic-ray research and provided for longer "staytimes."

It has been difficult to separate radiation effects from other space-flight factors: therefore, some of the alterations observed are still subject to debate. Vibration, acceleration, and weightlessness appear to be the three most important additional parameters. Measurements of radiation dosage have been made by chemical and photographic dosimetry, ion chambers, and biological dosimetry. All evidence to date indicates that radiation exposure levels are not hazardous to man at present orbital altitudes up to 200 nautical miles. Most biological materials flown so far have been for the express purpose of investigating space-radiation levels and effects. The biological materials have ranged from tissue cultures to entire organisms and from phage and bacterial cells to man. The studies have required much of the space and weight resources allotted biology by the U.S.S.R. and the United States. They have been accompanied by ground-based controls.

The Vostok series provided the following data:

(1) A small, but statistically significant, increase was observed in the percentage of chromosome aberrations in the rootlet cells of air-dried wheat and pea seeds after germination. In this case only, the increase did not depend on flight duration. (2) Lysogenic bacteria exhibited an increase of genetic alterations and increased phage production. Length of flight was associated with increased bacteriophage production by the lysogenic bacteria. There was an increase of recessive lethals coupled with nonconvergence of chromosomes (sex linked) in the fruit fly. A stimulation of cell division in wheat and pea seeds was observed. Cultures of human cells exposed to space-flight factors did not differ significantly from terrestrial controls with respect to such indicators as proliferation rate, percentage of mortality and morphological, antigenic, and cultural properties. Repeated flights of the identical HeLa cells revealed that there was a longer latent period for restoration of growth capacity than in cells carried into space once or not flown at all. (3) The most definite radiation effects observed were only revealed in genetic tests. No harmful influence on those characteristics affecting the viability of the organism has been discovered.

The Air Force Discoverer series launched from the west coast had a few successful flights incorporating organisms. With severe environmental stress and long recovery times, data on radiation exposure were equivocal up to Discoverer XVII and XVIII when cultures of human tissue were flown, recovered, and assessed for radiation exposure effects. Comparison with ground-based controls revealed no measurable differences.

Radiation dosimetry from the Mercury series established that minimal exposures were encountered at those orbital altitudes. A typical example is the MA-8 flight of W. M. Schirra, Jr., during which the body surface dosage was less than 30 millirads.

NASA has supported fundamental radiation studies at the Oak Ridge National Laboratory and the Lawrence Radiation Laboratory. Emphasis has been placed on the biological effects of high-energy proton radiation and particulate radiation from accelerators.

At the NASA Ames Research Center extensive fundamental studies are being carried out on the effects of radiation, especially in the nervous system. It has been demonstrated that deposits accumulate in the brain following exposure to large doses of ionizing particle radiation as well as after X-irradiation. These deposits, referred to as a "chemical lesion," result from an accumulation of glycogen. The formation of these deposits during exposure to large doses of X-irradiation was not increased in environments of 99.5 percent oxygen and increased atmospheric pressure.

SIMULATION OF PLANETARY (MARTIAN) ENVIRONMENTS

Attempts have been made to simulate to some degree the various parameters of the Martian environment, such as atmospheric composition, pressure, radiation flux, temperatures, and the day-night as well as seasonal cycles. Certain factors for Mars cannot yet be simulated, such as soil composition, gravitational field, magnetic field, and electrical field.

Caution is required in interpreting all simulation experiments. How Earth organisms respond to simulated Martian environments probably has nothing to do with life on Mars, but these experiments may show whether or not anything in the environment of Mars makes life as we know it impossible. We must expect that on Mars, life will have evolved and have adapted over long periods of time under conditions which are quite different from conditions on Earth. The simulation experiments also provide some information about the possibility of contaminating the planet Mars, or any planet, with organisms from Earth. In addition, they give us some clues about the possibilities of adaptation and evolution of life under these conditions.

From an evolutionary point of view, if life has developed on Mars, we expect it to have evolved at least to a microbial stage. On Earth, micro-organisms are the most ubiquitous and numerous forms of life. This fact should be considered in studying extraterrestrial bodies.

Micro-organisms have been selected as the best test organisms, and bacteria and fungi have been used because they are durable and easy to grow. Also, because of their rapid growth, many generations can be studied in a relatively short period of time. The organisms include chemoautotrophic bacteria, which are able to synthesize their cell constituents from carbon dioxide by energy derived from inorganic reactions; anaerobic bacteria, which grow only in the absence of molecular oxygen; photoautotrophic plants such as algae, lichens, and more complex seed plants; and small terrestrial animals.

Organisms have been collected from tundra, desert, hot springs, alpine, and saline habitats to obtain species with specialized capabilities to conserve water, balance osmotic discrepancies, store gases, accommodate to temperature extremes, and otherwise meet stresses. An attempt is made in these simulation experiments to extend these processes across the possible overlapping microenvironments which Earth and Mars may share.

Scientists have developed various special environmental simulators, including "Mars jars" and "Marsariums." These have made possible controlled temperatures, atmospheres, pressures, water activities, and soil conditions for duplicating assumed Martian surface. A complex simulator, developed by Young et al. ([ref.52]), reproduces the formation of a permafrost layer with some water tied up in the form of ice beneath the soil surface. This simulator serves as a model to study the wave of darkening, thus supporting the hypothesis that the pole-to-equator wave of darkening is correlated with the availability of subsurface water. The simulator is a heavily insulated 2-cu-ft capacity chamber with an internal pressure of 0.1 atm. The chamber contains a soil mixture of limonite and sand and an atmosphere of carbon dioxide and nitrogen. With the use of a liquid nitrogen heat exchanger at one end and an external battery of infrared lamps at the other end, the temperature simulates that of Mars from pole to equator. Thermocouples throughout the soil monitor the temperatures in the chamber.

Zhukova and Kondratyev ([ref.69]) designed a structure measuring 100×150×180 cm. Micro-organisms were placed at the surface of a copper bar made in a special groove separated by glass cloth. Copper was selected as one of the best heat-conduction materials permitting a rapid change of temperature. The lower end of the bar was immersed into a mixture of dry ice and ethyl alcohol, which made it possible to create a temperature of -60° C. Heating was performed by an incandescent spiral.

As the knowledge concerning the Martian environment becomes more refined, scientists can more accurately simulate this environment under controlled conditions in the laboratory. Determination of the effects of the Martian environment on Earth organisms will permit better theorization on the forms of life we might find on Mars and will permit us to estimate the potential survival of Earth contaminants on Mars.

However, until the environmental conditions of Mars are defined more accurately, the experiments must be changed continually to fit newly determined conditions. Therefore, existing simulation data are made less valid for comparison. The data resulting from the simulation experiments for Mars have been compiled in table II, and the experiments are summarized below.

The earliest simulation studies were carried out by the Air Force, and the studies during the past 6 years have been supported by NASA. Recently, these studies have received less support or have been terminated in favor of critical studies on the effects of biologically important environmental extreme factors on Earth organisms. These critical studies permit establishing the extreme environmental factor parameters in which Earth life can grow or survive. These data will have valuable application to the consideration of life on any planet, to the design of life-detection instruments, to the sterilization of space vehicles, and to the problem of contamination of planets.

Some exploratory experimental studies are in progress to study the capabilities of organisms to grow under the assumed conditions on Jupiter. These include studies at high pressure with liquid ammonia, methane, and other reducing compounds.

Early experiments simulating Martian conditions using soil bacteria were carried out by Davis and Fulton ([ref.70]) at the Air Force School of Aviation Medicine, San Antonio, Tex. Mixed populations of soil bacteria were put in "Mars jars" with the following conditions: 65-mm Hg pressure, 1 percent water or less, nitrogen atmosphere, sandstone-lava soil, and a temperature day-night cycle of +25° to -25° C. The moisture was controlled by desiccating the soil and adding a given amount of water. Experiments, conducted up to 10 months, demonstrated that obligate aerobes died quickly. The anaerobes and sporeformers survived. Although a small increase in the total number of organisms indicated growth, the increases in the number of bacteria may have been due to breaking up clumps of dirt.

Roberts and Irvine ([ref.71]) reported that, in a simulated Martian environment, colony counts of a sporeforming bacterium, Bacillus cereus, increased when 8 percent moisture was added. Moisture was considered more important than temperature or atmospheric gases inasmuch as a simulated Martian microenvironment containing 8 percent moisture permitted germination and growth of endospores of Clostridium sporogenes. Increases in colony counts of Bacillus cereus appeared to be influenced by temperature cycling ([ref.72]).

Table II.—Survival and Growth of Organisms in Simulated Planetary (Martian) Environments

------------------------------------------------------------------ Species Survival, Moisture Temperature, months °C

------------------------------------------------------------------ Conditions on Mars: 14µ±7µ -70 to +30 ------------------------------------------------------------------ Anaerobic 6 Low, -60 to +20 sporeformers (CaSO₄) Clostridia, Bacillus planosarcina ------------------------------------------------------------------ Anaerobic 6 Low, -60 to +20 nonsporeformers (CaSO₄) Pseudomonas, Rhodopseudomonas ------------------------------------------------------------------ Anaerobes Growth Very wet -75 to +25 Aerobacter aerogenes, Pseudomonas sp. ------------------------------------------------------------------ Clostridium, 10 1 -25 to +25 Corynebacteria percent "Thin short rod" or less ------------------------------------------------------------------ Bacillus cereus 2 0.5 -25 to +25 percent soil ------------------------------------------------------------------ Clostridium sporogenes 1 8.4 -25 to +25 (growth) percent ------------------------------------------------------------------ Clostridium botulinum 10 Lyophilized -25 to +25

------------------------------------------------------------------ Klebsiella pneumoniae 6 Lyophilized -25 to +25

------------------------------------------------------------------ Bacillus subtilis var. 4 2 percent -25 to +25 globigii ------------------------------------------------------------------ Sarcina aurantiaca 4 0.5 percent -25 to +25

------------------------------------------------------------------ Clostridium tetani 2 or less 1 percent -60 to +25 ------------------------------------------------------------------ Aspergillus niger Over 6 hr Very dry -60 to +25

------------------------------------------------------------------ Aspergillus oryzae Over 6 hr Very dry -60 to +25 ------------------------------------------------------------------ Mucor plumbeus Over 6 hr Very dry -60 to +25 ------------------------------------------------------------------ Rhodotorula rubra Over 6 hr Very dry -60 to +25 ------------------------------------------------------------------ Pea, bean, tomato, rye, 0.3 Moist +25 sorghum, rice. ------------------------------------------------------------------ Winter rye 0.6 Moist -10 to +23 ------------------------------------------------------------------

Table II.—Survival and Growth of Organisms in Simulated Planetary (Martian) Environments

---------------------------------------------------------------------- Species Atmospheric N₂, CO₂, Substrate pressure, percent percent mm Hg ---------------------------------------------------------------------- Conditions on Mars: 85, 3 to 30 25±15, 11 ---------------------------------------------------------------------- Anaerobic 76 95 5 Air-dried sporeformers soil Clostridia, Bacillus planosarcina ---------------------------------------------------------------------- Anaerobic 76 95 5 Air-dried nonsporeformers soil Pseudomonas, Rhodopseudomonas ---------------------------------------------------------------------- Anaerobes 760 100 (?) Difco Aerobacter infusion aerogenes, broth Pseudomonas sp. ---------------------------------------------------------------------- Clostridium, 65 100 (?) Soil Corynebacteria "Thin short rod" ---------------------------------------------------------------------- Bacillus cereus 65 94 2.21 Sandstone soil

---------------------------------------------------------------------- Clostridium sporogenes 65 94 2 Enriched soil ---------------------------------------------------------------------- Clostridium botulinum 65 95 0 to Lava soil 0.5 ---------------------------------------------------------------------- Klebsiella pneumoniae 65 95 0 to Lava soil 0.5 ---------------------------------------------------------------------- Bacillus subtilis var. 85 95 0.3 Media globigii ---------------------------------------------------------------------- Sarcina aurantiaca 85 95 0.3 Desert soil ---------------------------------------------------------------------- Clostridium tetani 85 95 0.3 Soil ---------------------------------------------------------------------- Aspergillus niger 76 95.5 0.25 Glass cloth on copper bar ---------------------------------------------------------------------- Aspergillus oryzae 76 95.5 0.25 Do. ---------------------------------------------------------------------- Mucor plumbeus 76 95.5 0.25 Do. ---------------------------------------------------------------------- Rhodotorula rubra 76 95.5 0.25 Do. ---------------------------------------------------------------------- Pea, bean, tomato, rye, 75 100 0 Filter sorghum, rice. paper ---------------------------------------------------------------------- Winter rye 76 98 0.24 Soil ----------------------------------------------------------------------

Studies of the effects of simulated Martian environments on sporeforming anaerobic bacteria were carried out by Hawrylewicz et al. ([ref.49]). They showed that the encapsulated facultative anaerobe, Klebsiella pneumoniae, survived under simulated Martian atmosphere for 6 to 8 months, but were less virulent than the freshly isolated organisms. Spores of the anaerobe Clostridium botulinum survived 10 months in the simulator. Hagen et al. ([ref.53]) found that the addition of moisture to dry-simulated Martian soil did not improve the survival of Bacillus subtilis or Pseudomonas aeruginosa. Bacillus cereus spores survived, with added organic medium plus moisture, but no germination of the spores resulted.

Hawrylewicz et al. ([ref.49]) put rocks from Antarctica bearing various lichens in simulated Martian conditions in a large desiccator. They found that the algal portion of a lichen, Trebouxia erici, showed only slight resistance to the Martian environment. They also pointed out the effect moisture had on the physical condition of lichens. The undersurface of a lichen has great water-absorbing capability, and the slightest amount of moisture on a rock surface is absorbed by the lichen which can turn green in 15 minutes.

Scher et al. ([ref.51]) exposed desert soils to simulated environmental conditions and diurnal cycles of Mars. The atmosphere consisted of 95 percent nitrogen and 5 percent carbon dioxide (no oxygen) and was dried, using calcium sulfate as a desiccant. The total atmospheric pressure was 0.1 atm. The temperature ranged from -60° to +20° C in 24-hour cycles. One hour was spent at the maximum and at the minimum temperatures. The chambers were irradiated with ultraviolet, 2537 Å, with a dose of 10⁹ ergs/cm², which is comparable to a daily dose found on Mars, and easily exceeds the mean lethal dose for unprotected bacteria. Soil aliquots were removed weekly and incubated at 30° C. The scoring was done both aerobically and anaerobically. Sporeforming obligate and facultative anaerobes, including Clostridium, Bacillus, and Planosarcina, and nonsporeforming facultative anaerobes, including Pseudomonas and Rhodopseudomonas, were found. The experimental chambers were frozen and thawed cyclically up to 6 months. Organisms that were able to survive the first freeze-thaw cycle were able to survive the entire experiment. The ultraviolet irradiation did not kill subsurface organisms, and a thin layer of soil served as an ultraviolet shield. All of the samples showed survivors.

Young et al. ([ref.52]) assumed that water is present on Mars, at least in microenvironments, and that nutrients would be available. The primary objective of their experiments was to determine the likelihood of contaminating Mars with Earth organisms should a space probe from Earth encounter an optimum microenvironment in terms of water and nutrients. The experiments used bacteria in liquid nutrient media. The environment consisted of a carbon dioxide-nitrogen atmosphere, and the temperature cycling was -70° to +25° C, with a maximum time above freezing of 4½ hours. Aerobacter aerogenes and Pseudomonas sp. grew in nutrient medium under Martian freezing and thawing cycles. Atmospheric pressure was not a significant factor in the growth of bacteria under these conditions.

Silverman et al. ([ref.47]) studied bacteria and a fungus under extreme—but not "Martian"—conditions. Spores of five test organisms (B. subtilis var. niger, B. megaterium, B. stearothermophilus, Clostridium sporogenes, and Aspergillus niger) and soils were exposed while under ultrahigh vacuum to temperatures of from -190° to +170° C for 4 to 5 days. Up to 25° C there was no loss in viability; at higher temperatures, differences in resistivity were observed. At 88° C, only B. subtilis and A. niger survived in appreciable numbers; at 107° C, only A. niger spores survived; none were recoverable after exposure to 120° C. B. subtilis survived at atmospheric pressure and 90° C for 5 days, but none of the other spores were viable alter 2 days. Four groups of soil organisms (mesophilic, aerobic, and anaerobic bacteria, molds, and actinomycetes) were similarly tested in the vacuum chamber. From one sample only actinomycetes survived 120° C, while one other soil sample yielded viable bacteria after exposure to 170° C. Several organisms resisted 120° C in ultrahigh vacuum for 4 to 5 days. When irradiated with gamma rays from a cobalt 60 source, differences were observed between vacuum-dried spores irradiated while under vacuum and those exposed to air immediately before irradiation. A reduction of from one-third to one-ninth of the viability of spores irradiated in vacuum occurred with vacuum-treated spores irradiated in air.

Siegel et al. ([ref.73]), in approximate simulations of Martian environments, studied tolerances of certain seed plants, such as cucumbers, corn, and winter rye, to low temperatures and lowered oxygen tensions. Lowered oxygen tensions enhanced the resistance of seedlings, particularly cucumber and rye to freezing, and lowered the minimum temperature required for germination. Germination of seeds in the absence of liquid water has also been studied. In this case, seeds of xerophytes have been suspended in air at 75-mm Hg pressure above water. The air was thus saturated. Germination was slow but did occur.

Siegel et al. (refs. [ref.73] and [ref.74]) found that the growth rate of several higher plants was enhanced by certain gases usually thought to be toxic, such as N₂O. This finding is significant inasmuch as the presence of nitrogen oxides in the Martian atmosphere has been cited as evidence for the nonexistence of plants on that planet by Kiess et al. ([ref.75]). Exploratory survival tests showed that various mature plants, as well as the larvae, pupae, and adult specimens of a coleopteran insect, were undamaged when exposed to at least 40 hours of an atmosphere containing 96.5 percent N₂O, 0.7 percent O₂, and 2.8 percent N₂.

Lichens are of interest because of their ability to survive and thrive under extreme environmental conditions on Earth. Biological activity of slow-growing lichens was detected by metabolic gas exchange, CO₂ detection being especially convenient. Siegel points out that this method is sensitive and nondestructive, to be preferred to staining techniques, which at present are limited because they are only semiquantitative, subjective, and destructive of the lichen.

A Russian study of simulated planetary environments has been performed with good simulation but for periods of only 2 to 6 hours. Comments on simulation experiments made by Zhukova and Kondratyev ([ref.69]) are presented as follows:

On the basis of modern conceptions on Martian conditions it is difficult to imagine that higher forms of animals or plants exist on the planet. A Martian change of seasons similar to that of our planet empowers us to think that there is a circulation of an organic substance on Mars, which cannot exist without participation of microbic forms of life. Microorganisms are the most probable inhabitants of Mars although the possibility is not excluded that their physiological features will be very specific. That is why the solution of the problem concerning the character of life on Mars is of exceptional interest. But still the answer to this question can be verified only by simulating Martian conditions, taking into account the information obtained from astrophysicists.

Experiments aimed at creating artificial Martian climatic conditions have been started quite recently; their number is not large since they cannot be combined with the results of numerous experiments investigating the effect of extreme factors on microorganisms. The result of the effect of such physicochemical parameters of the medium as pressure, sharp temperature changes, the absence of oxygen and insolation, depends on their combination and simultaneity. These examples convincingly show that while simulating Martian conditions one should strive to the most comprehensive complex of simultaneously acting factors. The creation of individual climatic parameters acting successively leads to absolutely different, often opposite results. It should be mentioned also that refusal to imitate insolation and the performance of experiments with specimens of soil which itself has protective effect on cells of microorganisms, but not with pure culture of bacteria, are usual shortcomings in the bulk of studies on this problem.

It appears that organisms from Earth might survive in large numbers when introduced to Martian environment. Whether these organisms will be capable of growth and explosive contamination of the planet in a biological sense or not is highly questionable. The likelihood of an organism from Earth finding ideal conditions for growth on Mars seems extremely low. However, the likelihood of an organism from Earth serving as a contaminant for any life-detection device flown to Mars for the purpose of searching out carbon-based life is considerably higher. The chance that life has originated and evolved on Mars is a completely separate question and much more difficult to answer.

It would be interesting to attempt to determine possible evolutionary trends which might occur on a planet by means of selection of organisms in a simulated planetary environment. Rapid genetic selection combined with radiation and chemicals to speed up mutation rate under these conditions should reveal possible evolutionary trends under the planetary environmental conditions. This could be attempted after the planetary environments are more accurately defined.

EXTREME AND LIMITING ENVIRONMENTAL PARAMETERS OF LIFE

The question of the existence of extraterrestrial life is one of the most important and interesting biological questions facing mankind and has been the subject of much controversial discussion and conjecture. Many of the quantitative, and even qualitative, environmental constituents of the planets also are still subjects of controversy and speculation. Best guesses about a relatively unknown planetary environment, combined with lack of information about the capabilities of Earth life to grow in extreme environments, do not provide the basis for making informed scientific estimates.

Life on Earth is usually considered to be relatively limited in its ability to grow, reproduce, or survive in extreme environmental conditions. While many common plants and animals (including man) are quite sensitive to, or incapable of, surviving severe chemical and physical changes or extremes of environment, a large number of micro-organisms are highly adapted and flourish in environments usually considered lethal. Certain chemoautotrophic bacteria require high concentrations of ammonia, methane, or other chemicals to grow. Anaerobic bacteria grow only in the absence of oxygen.

Besides adapting to the extremes of environments on Earth, life is also capable of growing and reproducing under extreme environmental conditions not normally encountered: e.g., from a few rad of radiation in normal habitats to 10⁶ or more rad from artificial sources, from 0.5 gauss of Earth magnetism to 167 000 gauss in manmade magnetic fields, and from 1-g force of gravity to 110 000 g. The extreme ranges of physical and chemical environmental factors for growth, reproduction, and survival for Earth micro-organisms are phenomenally large.

Life is ubiquitous on Earth and is found in almost every possible environment, including the most severe habitats, from the bottom of the ocean to the highest mountain tops and from cold Arctic habitats to hot springs, as well as in volcanic craters, deep wells, salt flats, and mountain snowfields. Earth life has become adapted to, and has invaded, nearly every habitat, no matter how severe. The physiological and morphological adaptations of life are exceedingly diverse and complex.

Surprisingly, the extreme parameters or ranges of the physical and chemical environmental factors permitting growth, reproduction, and other physiological processes of Earth organisms have not been critically compiled. A partial compilation of certain selected environmental factors has been made by Vallentyne ([ref.76]). A compilation of available published data on certain environmental extremes, particularly from recent NASA-supported research (compiled by Dale W. Jenkins, in press), is presented in tables III to VI. These data can serve as a starting point for a more intensive literature review by specialists, critical evaluation, standardization of end points, and especially to point out areas where critical experimentation is urgently needed.

This critical compilation involves a review of a very broad and complex range of subjects involved in many different disciplines with widely scattered literature. Since the effects of many of the specific environmental factors are harmful, it is difficult to select a point on a scale from no effect to death and use some criteria to say that normal or even minimal growth and reproduction are occurring. The effects of environmental factors are dependent on (1) the specific factor, times, (2) the concentration or energy, times, (3) the time of exposure or application of the factor. Many reports, especially older ones, do not give all of the necessary data to permit proper evaluation. A complicating factor is that the effect of each factor depends on the other factors before, during, and after its application. The condition of the organism itself is a great variable. Proper evaluation requires the critical review by a variety of biological specialists, physicists, and chemists.

To determine the potential of Earth organisms to survive or grow under other planetary environmental conditions, a number of experiments have been carried out attempting to simulate planetary environments, especially of Mars, as reviewed previously. While the results are of real interest, they do not provide much basic information. Further, as the Martian environment is more accurately defined, the experimental conditions are changed. In addition, some experimenters have altered certain factors, such as water content, to allow for potential microhabitats or for areas which might contain more water at certain times.

Table III.—Extreme Physical Environmental Factors

----------------------------------------------------------------- Physical Minimum Organism factors ----------------------------------------------------------------- Temperature -30° C Algae (photosynthesis), pink yeast (growth) ----------------------------------------------------------------- Magnetism 0-50 gamma (=×10⁻⁵ Human gauss)

----------------------------------------------------------------- Gravity 0 g Human, plants, animals

----------------------------------------------------------------- Pressure 10⁻⁹ mm Hg (5 days) Mycobacterium smegmatis ----------------------------------------------------------------- Microwave 0 W/cm²

----------------------------------------------------------------- Visible 0 ft-c Animals, fungi, bacteria

----------------------------------------------------------------- Ultraviolet 0 erg/cm²

----------------------------------------------------------------- X-ray 0 rad ----------------------------------------------------------------- Gamma ray 0 rad

----------------------------------------------------------------- Acoustic 0 dyne/cm²

-----------------------------------------------------------------

Table III.—Extreme Physical Environmental Factors

---------------------------------------------------------------------- Physical Maximum Organism Activity factors ---------------------------------------------------------------------- Temperature 104° C (1000 Desulfovibrio Grows and reduces atm) desulfuricans sulfate ---------------------------------------------------------------------- Magnetism 167 000 Neurospora 1 hr—no effect, gauss Arbacia Arbacia Drosophila development delayed ---------------------------------------------------------------------- Gravity 400 000 g Ascaris eggs 1 hr—eggs hatch, 40 110 000 g Escherichia coli days’ growth ---------------------------------------------------------------------- Pressure 1400 atm Marine organisms Growth

---------------------------------------------------------------------- Microwave 2450 Mc/sec Drosophila 68 hr, growth not 0.3 to 1 affected W/cm² ---------------------------------------------------------------------- Visible 50 000 ft-c Chlorella, Seconds, 17 000 ft-c higher plants recurrently continuous ---------------------------------------------------------------------- Ultraviolet 10⁸ erg/cm², Bean embryos Suppressed growth 2537 Å ---------------------------------------------------------------------- X-ray 2×10⁶ rad Bacteria Growth ---------------------------------------------------------------------- Gamma ray 2.45×10⁶ rad Microcoleus Continued growth Phormidium Synechococcus ---------------------------------------------------------------------- Acoustic 140 db or Man Threshold of pain 6500 dyne/cm² at 0.02 to 4.8 kcs/sec ----------------------------------------------------------------------

Table IV.—Extreme Low and High Temperature Effects Permitting Life Processes

----------------------------------------------------------------- Minimum Organism Activity or condition temperature, °C ----------------------------------------------------------------- -11 Bacteria Growth (on fish) ----------------------------------------------------------------- -12 Bacteria Growth ----------------------------------------------------------------- -12 Molds Growth ----------------------------------------------------------------- -15 Pyramidomonas Swimming ----------------------------------------------------------------- -15 Dunaliella salina Swimming ----------------------------------------------------------------- -18 Mold Growth ----------------------------------------------------------------- -18 Yeast Growth ----------------------------------------------------------------- -18 Aspergillus Growth (in glycerol) glaucus ----------------------------------------------------------------- -18 to -20 Mold Growth (in fruit juice) ----------------------------------------------------------------- -18 to -20 Pseudomonads Growth (in fruit juice) ----------------------------------------------------------------- -20 Bacteria Growth ----------------------------------------------------------------- -20 Bacteria Growth ----------------------------------------------------------------- -20 Bacteria Luminescence development accelerated ----------------------------------------------------------------- -20 to -24 Insect eggs (diapause) ----------------------------------------------------------------- -30 Algae Photosynthesis ----------------------------------------------------------------- -30 Pink yeast Growth (on oysters) ----------------------------------------------------------------- -30 Lichens Photosynthesis ----------------------------------------------------------------- -20 to -40 Lichens and conifers Photosynthesis ----------------------------------------------------------------- -44 Mold spores Sporulation and germination -----------------------------------------------------------------

Table IV.—Extreme Low and High Temperature Effects Permitting Life Processes

------------------------------------------------------------------ Maximum Organism Activity or condition temperature, °C ------------------------------------------------------------------ 73 Thermophilic organisms Growth (P³² metabolism) ------------------------------------------------------------------ 73 Phormidium (alga) Acclimatized ------------------------------------------------------------------ 70 to 73 Bacillus calidus Growth and spore germination ------------------------------------------------------------------ 70 to 74 Bacillus cylindricus Growth and spore germination ------------------------------------------------------------------ 70 to 75 Bacillus tostatus Growth and spore germination ------------------------------------------------------------------ 80 Bacillus Cultured in laboratory stearothermophilus ------------------------------------------------------------------ 83 Sulfate-reducing Found in a well bacteria ------------------------------------------------------------------ 89 Sulfate-reducing Found in oil waters bacteria ------------------------------------------------------------------ 65 to 85 Sulfate-reducing Cultured in laboratory bacteria ------------------------------------------------------------------ 89 Micro-organisms Found in hot springs ------------------------------------------------------------------ 95 Bacillus coagulans In 80 min. sporulation activation ------------------------------------------------------------------ 110 Bacillus coagulans In 6 min, sporulation activation ------------------------------------------------------------------ 104 Desulfovibrio Grow and reduce sulfate desulfuricans at 1000 atm ------------------------------------------------------------------

Table V.—Extreme Temperature Limits of Survival

-------------------------------------------------- Minimum Organism temperature °C -------------------------------------------------- -190 Yeast bacteria, 10 species -------------------------------------------------- -197 Trebouxia erici from lichens -------------------------------------------------- -197 Protozoa, Anguillula -------------------------------------------------- -252 Yeasts, molds, bacteria, 10 species -------------------------------------------------- -253 Black currant, birch -------------------------------------------------- -273 Bacteria, many species -------------------------------------------------- -273 Bacteria, many species -------------------------------------------------- -272 Desiccated rotifers -------------------------------------------------- -269 Human spermatozoa --------------------------------------------------

Table V.—Extreme Temperature Limits of Survival

------------------------------------------------------------------ Maximum Organism Time of exposure temperature °C ------------------------------------------------------------------ 140 Bacterial spores 5-hr immersion ------------------------------------------------------------------ 170-200 Desiccated rotifers 5 min ------------------------------------------------------------------ 151 Desiccated rotifers 35 min ------------------------------------------------------------------ 150 Clostridium tetani 180 min ------------------------------------------------------------------ 170 Aerobic bacteria, molds. 5 days at actinomycetes 6×10⁻⁹mm Hg ------------------------------------------------------------------ 127 (dry) Bacteria (in activated charcoal) 60 min ------------------------------------------------------------------ 110 (wet) Bacillus subtilis var. niger 400 min ------------------------------------------------------------------ 120 Bacillus subtilis var. niger 400 min ------------------------------------------------------------------ 141 Bacillus subtilis var. niger 70 min ------------------------------------------------------------------ 160 Bacillus subtilis var. niger 15 min ------------------------------------------------------------------ 180 Bacillus subtilis var. niger 2 min ------------------------------------------------------------------ 188 Bacillus subtilis var. niger 1 min ------------------------------------------------------------------ 120 (wet) Bacillus stearothermophilus 25 min ------------------------------------------------------------------ 120 (dry) Bacillus stearothermophilus 100 min ------------------------------------------------------------------ 141 Bacillus stearothermophilus 12 min ------------------------------------------------------------------ 160 Bacillus stearothermophilus 2 min ------------------------------------------------------------------ 166 Bacillus stearothermophilus 1 min ------------------------------------------------------------------

Table VI.—Extremes of Chemical Environmental Factors Permitting Growth or Activity

-------------------------------------------------------- Chemical Minimum Organism factor -------------------------------------------------------- O₂ 0% HeLa cells, Cephalobus, anaerobic bacteria -------------------------------------------------------- O₃ (ozone) 0%

-------------------------------------------------------- H₂ 0%

-------------------------------------------------------- H₂O Aw 0.48 Pleurococcus vulgaris ------------------------------------------ Aw 0.5 Xenopsylla cheopis (prepupae) -------------------------------------------------------- H₂O₂ 0%

-------------------------------------------------------- He 0%

-------------------------------------------------------- CO 0%

-------------------------------------------------------- CO₂ 0%

-------------------------------------------------------- CH₄ 0% -------------------------------------------------------- CH₂O 0% -------------------------------------------------------- CH₃OH 0% -------------------------------------------------------- N₂ 0%

-------------------------------------------------------- NO 0%

-------------------------------------------------------- NO₂ 0%

-------------------------------------------------------- N₂O 0%

-------------------------------------------------------- Ar 0% -------------------------------------------------------- NaCl, Na₂SO₄, NaHCO₃ -------------------------------------------------------- H₂S 0%

-------------------------------------------------------- H₂SO₄ 0%

-------------------------------------------------------- Cu⁺⁺

-------------------------------------------------------- Zn⁺⁺

-------------------------------------------------------- pH 0 Acontium velatum Thiobacillus thioodixans

-------------------------------------------------------- Eh -450 mV Sulfate-reducing bacteria at pH 9.5 --------------------------------------------------------

Table VI.—Extremes of Chemical Environmental Factors Permitting Growth or Activity

---------------------------------------------------------------------- Chemical Maximum Pressure, Time, Organism Activity factor atm days ---------------------------------------------------------------------- O₂ 100% 1 Plants, Growth animals ---------------------------------------------------------------------- O₃ 100 ppm 5 Armillaria Growth (ozone) --------------------------- mellea ----------------- 500 ppm 5 Light emission ---------------------------------------------------------------------- H₂ 100% Various Germination plants ---------------------------------------------------------------------- H₂O Aw 1.0 1 Various Growth aquatic organisms

---------------------------------------------------------------------- H₂O₂ 0.34% Rye Germination enhanced ---------------------------------------------------------------------- He 100% Wheat, rye, Germination rice ---------------------------------------------------------------------- CO 100% Rye Germination -------------------------------------------------------------- 80% 1.1 4 Hydrogenomonas Growth ---------------------------------------------------------------------- CO₂ 100% 1.1 4 Rye Growth and germination ---------------------------------------------------------------------- CH₄ 100% 1.1 4 Rye Germination ---------------------------------------------------------------------- CH₂O 50% Rye Germination ---------------------------------------------------------------------- CH₃OH 50% Rye Germination ---------------------------------------------------------------------- N₂ 100% .1 10 Various plants Germination and root growth ---------------------------------------------------------------------- NO 18% .018 10 Sorghum, rice Germination and root growth ---------------------------------------------------------------------- NO₂ 18% .018 10 Rye, rice Germination and root growth ---------------------------------------------------------------------- N₂O 100% 1.2 4 Rye Germination -------------------------------------------------------------- 96.5% 1.7 Rye Germination ------------------------------------ Tenebrio Survival molitor ---------------------------------------------------------------------- Ar 100% 1.2 2 Rye Germination ---------------------------------------------------------------------- NaCl, 67% Photosynthetic Growth Na₂SO₄, bacteria NaHCO₃ ---------------------------------------------------------------------- H₂S 0.96 Desulfovibrio Growth g/liter desulfuricans ---------------------------------------------------------------------- H₂SO₄ 7% Acontium Growth velatum ------------------------------- Thiobacilli Growth, reproduction ---------------------------------------------------------------------- Cu⁺⁺ 12 Thiobacillus Growth g/liter ferrooxidans ---------------------------------------------------------------------- Zn⁺⁺ 17 Thiobacillus Growth g/liter ferrooxidans ---------------------------------------------------------------------- pH 13 Plectonema Growth nostocorum ------------------------------- Nitrobacter Growth ------------------------------- Nitrosomonas Growth ---------------------------------------------------------------------- Eh 850 mV Iron bacteria Growth at pH 3 ----------------------------------------------------------------------